The Green Tea Polyphenol Epigallocatechin-Gallate (EGCG) Interferes with Microcin E492 Amyloid Formation.

Microcin E492 (MccE492) is an antimicrobial peptide and proposed virulence factor produced by some Klebsiella pneumoniae strains, which, under certain conditions, form amyloid fibers, leading to the loss of its antibacterial activity. Although this protein has been characterized as a model functional amyloid, the secondary structure transitions behind its formation, and the possible effect of molecules that inhibit this process, have not been investigated. In this study, we examined the ability of the green tea flavonoid epigallocatechin gallate (EGCG) to interfere with MccE492 amyloid formation. Aggregation kinetics followed by thioflavin T binding were used to monitor amyloid formation in the presence or absence of EGCG. Additionally, synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) were used to study the secondary structure, thermal stability, and morphology of microcin E492 fibers. Our results showed that EGCG significantly inhibited the formation of the MccE492 amyloid, resulting in mainly amorphous aggregates and small oligomers. However, these aggregates retained part of the ß-sheet SRCD signal and a high resistance to heat denaturation, suggesting that the aggregation process is sequestered or deviated at some stage but not completely prevented. Thus, EGCG is an interesting inhibitor of the amyloid formation of MccE492 and other bacterial amyloids.

Assessment of Intracellular Amyloid Formation in Fixed and Live Bacteria Using Fluorescence Microscopy.

Although amyloid aggregation has been generally associated with protein misfolding and neurodegenerative diseases in mammals, bacteria and other organisms have harnessed amyloidogenesis to perform diverse biological processes. These functional amyloids, some of them secreted and others intracellular, require that the producing cells keep aggregation under control in the cytoplasm upon protein translation, preventing their inherent toxicity. Thus, it is highly relevant to understand how intracellular amyloid formation occurs and is regulated, its metabolic consequences, and the formation dynamics and fate of the amyloid inclusions upon cell division. This chapter describes methods leveraging fluorescence microscopy and fixed- or live-cell imaging to monitor intracellular amyloid formation in bacterial cells.

Identification of Aggregation-Prone and Gatekeeper Residues in Bacterial Amyloids Using Site-Directed Mutagenesis and Flow Cytometry.

Bacterial functional amyloids are remarkable examples of how amyloid aggregation can be kept under control and even leveraged to perform diverse biological processes. In this context, it is highly relevant to understand how amyloidogenesis is modulated by relevant factors, including key amino acids promoting or preventing aggregation. This chapter describes a methodology to identify critical residues for amyloid formation in bacterial proteins, based on mutant construction guided by bioinformatics prediction, their expression in bacteria, and their analysis by flow cytometry. Additionally, we describe a simple downstream analysis of selected mutants to assess their in vitro aggregation properties upon protein purification. We applied the proposed methodology to identify critical residues modulating the aggregation of the antimicrobial peptide microcin E492, a well-studied model of bacterial amyloids.

CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev.

Translation initiation of the human immunodeficiency virus type-1 (HIV-1) full-length RNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the full-length RNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF inhibits HIV-1 and HIV-2 Gag synthesis from the full-length RNA. Our results indicate that CTIF associates with HIV-1 Rev through its N-terminal domain and is recruited onto the full-length RNA ribonucleoprotein complex in order to interfere with Gag synthesis. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that Rev interferes with the association of CTIF with CBP80 indicating that CTIF and Rev compete for the CBC subunit.

Relevant Genes Linked to Virulence Are Required for Salmonella Typhimurium to Survive Intracellularly in the Social Amoeba Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum has proven to be a useful model for studying relevant aspects of the host-pathogen interaction. In this work, D. discoideum was used as a model to study the ability of Salmonella Typhimurium to survive in amoebae and to evaluate the contribution of selected genes in this process. To do this, we performed infection assays using axenic cultures of D. discoideum co-cultured with wild-type S. Typhimurium and/or defined mutant strains. Our results confirmed that wild-type S. Typhimurium is able to survive intracellularly in D. discoideum. In contrast, mutants ΔaroA and ΔwaaL are defective in intracellular survival in this amoeba. Next, we included in our study a group of mutants in genes directly linked to Salmonella virulence. Of note, mutants ΔinvA, ΔssaD, ΔclpV, and ΔphoPQ also showed an impaired ability to survive intracellularly in D. discoideum. This indicates that S. Typhimurium requires a functional biosynthetic pathway of aromatic compounds, a lipopolysaccharide containing a complete O-antigen, the type III secretion systems (T3SS) encoded in SPI-1 and SPI-2, the type VI secretion system (T6SS) encoded in SPI-6 and PhoP/PhoQ two-component system to survive in D. discoideum. To our knowledge, this is the first report on the requirement of O-antigen and T6SS in the survival of Salmonella within amoebae. In addition, mutants ΔinvA and ΔssaD were internalized in higher numbers than the wild-type strain during competitive infections, suggesting that S. Typhimurium requires the T3SS encoded in SPI-1 and SPI-2 to evade phagocytosis by D. discoideum. Altogether, these results indicate that S. Typhimurium exploits a common set of genes and molecular mechanisms to survive within amoeba and animal host cells. The use of D. discoideum as a model for host-pathogen interactions will allow us to discover the gene repertoire used by Salmonella to survive inside the amoeba and to study the cellular processes that are affected during infection.

Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation.

Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril's morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4'-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54-63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54-63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts.

Estilo de vida e índice de masa corporal de una población de adultos del sur de Tamaulipas, México / Lifestyle and Body Mass Index of an Adult Population in Southern Tamaulipas, Mexico / Estilo de vida e índice de massa corporal de uma população adulta no sul de Tamaulipas, México

La obesidad es una enfermedad multifactorial que involucra aspectos genéticos y ambientales. Objetivo: valorar la influencia del estilo de vida en el índice de masa corporal de una población de adultos. Método: este estudio descriptivo transversal se deriva del proyecto "Hábitos alimentarios y composición corporal (IMC) de adultos con sobrepeso y obesidad"; cuya fase de recolección se realizó entre enero y julio de 2009; preliminarmente se presentan resultados relativos al estilo de vida. Se aplicó una cédula de datos personales y se realizaron mediciones antropométricas. Para el análisis estadístico se utilizaron medidas de tendencia central y se construyeron modelos de regresión lineal múltiple. Resultados: las mayores proporciones tanto del grupo de hombres como del de mujeres, señalaron no fumar, ni realizar algún deporte o ejercicio. En el caso del consumo de alcohol, la mayor proporción del grupo de hombres dijo consumirlo. Los modelos de regresión mostraron que en el total del grupo, así como en el grupo de mujeres, las variables tabaco y ejercicio influyeron en el IMC de los participantes, ya que tuvieron efecto en el modelo de regresión. Dichas variables se correlacionaron significativa e inversamente con el IMC, lo que particularmente llamó la atención en el caso del tabaco. Discusión: el estudio muestra que el estilo de vida influye de manera importante en el IMC de esta población de adultos.

Obesity is a multifactor disease involving genetic and environmental aspects. Objective: Assess the influence of lifestyle on the body mass index (BMI) of an adult population. Method: This descriptive study is derived from a project entitled The Eating Habits and Body Composition of Overweight and Obese Adults. The data collection phase was from January to July 2009, and the preliminary results are related to lifestyle. A format for collecting personal data was applied and anthropometric measurements were taken. For the statistical analysis, central tendency measurements were used and multiple linear regression models were constructed. Results: The largest proportions of both groups - men and women - indicated they neither smoke, nor practice sports or exercise. In the case of alcohol consumption, the largest proportion of the male group indicated alcohol consumption. The regression models showed that, in the group of participants as a whole and in the female group, the tobacco and exercise variables influenced the BMI, inasmuch as they had an effect on the regression model. These variables were significant and inversely correlated with the BMI, particularly in the case of tobacco. Discussion: The study shows that lifestyle plays an important role in the BMI of the adult population.

A obesidade é uma doença multifatorial que envolve aspectos genéticos e ambientais. Objetivo: avaliar a influência do estilo de vida sobre o índice de massa corporal de uma população adulta. Método: estudo descritivo transversal derivado do projeto "Hábitos alimentares e composição corporal de adultos com sobrepeso e obesidade", cuja fase de coleta foi realizada entre janeiro e julho de 2009. Os resultados preliminares estão relacionados ao estilo de vida. Se registraram os dados pessoais e as medidas antropométricas. A análise estatística utilizou medidas de tendência central e construiu vários modelos de regressão linear. Resultados: A maioria de ambos os grupos de homens e mulheres disse não fumar nem praticar esportes ou exercícios. A maior proporção de homens consume álcool. Os modelos de regressão mostraram que, na totalidade do grupo e no grupo de mulheres as variáveis cigarro e exercício influenciaram o IMC dos participantes, posto que tiveram efeito sobre o modelo de regressão. Essas variáveis se correlacionaram significativamente e inversamente com o IMC, o que chamou a atenção em especial no caso do cigarro. Discussão: o estudo mostra que o estilo de vida desempenha um papel importante no IMC desta população adulta.

Niveles de bienestar espiritual y de fortaleza relacionados con la salud en adultos mayores / Levels of spiritual well-being and strength related to the health in greater adults

Este estudio investigó la relación entre niveles de bienestar espiritual y fortaleza relacionada con la salud en una población mexicana de adultos mayores de 65 años. El proyecto se basó en el Modelo de Adaptación de Roy, el Modelo de Pollock (fortaleza relacionada con la salud) y el de Reed (bienestar espiritual). El diseño fue descriptivo correlacional, la muestra fue probabilística y se hizo al azar. La muestra (n=160) tuvo un nivel de significancia de 0,05 para una diferencia media de 1,6, un efecto de tamaño y potencia de 80. Los instrumentos fueron la Escala de Fortaleza Relacionada con la Salud, y la de Bienestar Espiritual, y presentaron un Alfa de 0,801 y 0,973 respectivamente. Se utilizó la Regresión Lineal Múltiple para investigar el efecto de las variables demográficas sobre el bienestar espiritual. La edad media de los sujetos fue de 73,9 (DE=4,3), el 62,5 porciento fueron mujeres. El coeficiente de correlación entre el bienestar espiritual y la fortaleza relacionada con la salud fue significativo, los niveles más altos de bienestar espiritual tienen niveles igualmente elevados de fortaleza relacionada con la salud. El sexo, la edad, la educación y el estado civil mostraron una relación positiva con los niveles de bienestar espiritual (p<,005). Las mujeres mostraron un nivel mayor de bienestar espiritual que los hombres